Project Summary Breast cancer brain metastases (BCBM) affect up to half of patients with advanced HER2+ breast cancer and 10-15% of patients with advanced ER+/HER2- breast cancer. Standard of care includes surgery and/or radiotherapy; however, these approaches can be associated with substantial toxicities, do not address systemic disease, and leave patients at risk for future central nervous system progression (CNS) and death. Despite their clinical impact, existing preclinical models of BCBM have been limited, and the factors which influence BCBM growth are not well elucidated. To date, no systemic therapy has gained regulatory approval for the treatment of BCBM?hence this represents an area of major, persistent, and unmet medical need. Preclinical investigations, including our own, have suggested a role for at least two key pathways? PI3K/PTEN/mTOR and cyclin D1/CDK4?in the growth and maintenance of BCBM. The overarching goals of this project are to elucidate the roles of the PI3K/PTEN/mTOR pathway and the Cyclin D1/CDK4 pathway in the growth and development of BCBM, to dissect the basis of site-specific response/resistance to inhibitors of these pathways, to test the clinical utility of targeting the pathways in patients with BCBM, and to identify ways to predict and overcome therapeutic resistance, with the long-term goal of identifying more effective treatment and prevention strategies. To accomplish our aims, we have assembled a multidisciplinary team enabling close bi-directional collaboration between the laboratory and clinic. We will leverage our unique collection of patient-derived xenograft (PDX) models generated from human BCBM specimens, and genetically-engineered mouse models (GEMMs), married with state-of-the art molecular pathology techniques. In Aim 1, we will 1) test whether PTEN loss promotes the growth and maintenance of BCBMs, and evaluate the effects of genetic or pharmacologic restoration of PTEN expression; 2) evaluate brain-penetrant PI3K/mTOR inhibitors in preclinical models of BCBM and uncover potential mechanisms of site-specific resistance; and 3) test the efficacy of combined PI3K/mTOR blockade in patients with HER2+ BCBM. In Aim 2, we will 1) evaluate the efficacy of CDK4/6 inhibition, alone and in rational combinations, 2) evaluate the efficacy and immuno-modulatory effects of CDK4/6 inhibitors, alone and in combination with immune checkpoint blockade and in varying genetic backgrounds, and 3) explore the clinical efficacy of combined HER2 and CDK4/6 inhibition in patients with HER2+ BCBM. Together, these studies will further our understanding of the pathophysiology of BCBM, strengthen our ability to overcome therapeutic resistance, and improve outcomes for patients with this disease.